This project has focused on development and application of technology to evaluate signaling events in the laboratory and in clinical and preclinical samples. Work is nearly complete on the application of the reverse phase protein array (RPPA) to surrogate measurements of angiogenesis. These studies have applied the marker CD31/PECAM to endothelial cell culture lysates and archival anonymized tumor samples. A cross validation against counts of CD31-stained microvessels is pending at this time. Success with this approach will provide a relative measurement of vascularity from as little as a single slice of tumor or triplet of slices of a core needle biopsy, of the type taken in our clinical trials, and will be applied to an ongoing prospectively accruing trial. Related to this are studies to optimize and validate the RPPA for further application to clinical samples. The focus of the last year has been on quality assurance and control of the pixelation and analysis of the RPPA results and more recently optimization of the lysis buffer used for tissue samples and total protein staining for loading control. Studies with CAI remain collaborative, recent results assessing CAI in imatinib-resistant CML cells is promising. CAI treatment inhibited activation of abl kinase and MAPK in these cells. Use of CAI for ophthalmologic disease has been licensed and proceeds with collaboration. CAI will be tested therapeutically for benefit in macular degeneration. This comprehensive approach to angiogenesis and microenvironment study in the laboratory and patient samples will provide opportunity for proof of principle of molecularly targeted agents in stromal therapy and will complement our continuing clinical work with signal transduction inhibition therapy.